Method of continuous automatic incubation of bacteria and determination of growth thereof, and apparatus therefor

ABSTRACT

A method and a means for the culture of bacteria with or without shaking, in which supply of culture media and test material, the stages of inoculation, and periodic determination of the growth of bacteria are accomplished in a continuous, automatic process.

United States Patent 1 Chibata et al.

[ Dec. 16, 1975 METHOD OF CONTINUOUS AUTOMATIC INcUBATIoN 0F BACTERIA AND DETERMINATION OF GROWTH THEREOF, AND APPARATUS THEREFOR [75] Inventors: lchiro Chibata, Suita; Hiroshi Ito,

ltami; Tomoaki Morimoto, Suita', Yukio Taniguchi, Toyonaka; Tsuneo Fujimoto, Amagasaki, all of Japan [73] Assignee: Tanabe Seiyaku Co., Ltd., Osaka,

Japan 22 Filed: Sept. 21, 1973 21 Appl. No.: 399,309

[30] Foreign Application Priority Data Sept. 27, 1972 Japan 47-96734 Sept. 27, 1972 Japan 47-96735 [52] US. Cl. 195/103.5; 195/115', 195/139; 195/142 [51] Int. Cl. C12B 1/00 [58] Field of Search. 195/103.5, 127, 139, 140-142, 195/115 [56] References Cited UNITED STATES PATENTS [57] ABSTRACT A method and a means for the culture of bacteria with or without shaking, in which supply of culture media and test material, the stages of inoculation, and periodic determination of the growth of bacteria are accomplished in a continuous, automatic process.

5 Claims, 16 Drawing Figures US. Patent Dec. 16, 1975 Sheet 1 of7 3,926,733

; FIG. 2

25 31 so 2%: 24b

US. Patent Dec. 16, 1975 Sheet20f7 3,926,733

FIG. 3

Sorbose produced m a o 0 Angutur dusplacemen? Impact frequancy US. Patent Dec. 16, 1975 Sheet 3 of? 3,926,733

US. Patent Dec. 16, 1975 Sheet 5 of7 3,926,733

FlG. IO

FIG.

US. Patent Dec. 16, 1975 Sheet60f7 3,926,733

FIG. I4

R FIG. l2 sosb I050 m5 105 I051:

78a 78b 78 78c FiG. i5

US. Patent Dec. 16, 1975 Sheet7of7 3,926,733

FIG. l5

FIG.|6

Convention O 5 0mm 5 2 6230 L- isoleucine (,uq/ml) METHOD OF CONTINUOUS AUTOMATIC INCUBATION OF BACTERIA AND DETERMINATION OF GROWTH THEREOF, AND

APPARATUS THEREFOR The present invention relates to a method for culturing bacteria with shaking and to an apparatus employed in said method, and more particularly to an improved method and means for providing a good air supply to aerobic microorganisms to accelerate the growth thereof.

Microorganisms are frequently employed in the development or production of medical or chemical compounds, or for microbioassay of amino accids and vitamins as well as for test for activities of antibiotics. In order to promote the growth of aerobic microorganisms it has been known to incubate the microorganisms in a medium with a constant supply of oxygen, which is commonly effected by shaking the culture and the medium. In conventional shaking methods and means, cultures may be placed in test-tubes which are then shaken or rotated. Conventional methods and means have the disadvantage that if the test-tubes are held vertically, oxygen supply to the cultures is poor, since, in a test-tube, the surface area exposed to the air is small, and even if the test-tubes are inclined in order to increase this surface area, the amount of oxygen moving into the cultures still remains small. When attempts are made to overcome this problem by vigorous shaking of the test-tubes, there is frequently leakage and loss of culture broth past stoppers.

In addition, processes involved in the microbiological growth experiments are injection of a culture medium and sample solutions, inoculation, incubation, and determination of the bacterial growth. Because of the complexity of the above-described processes, it is highly desirable that the processes be automated as fully as possible. However, there is no overall system with a degree of automation permittingresearch to advance smoothly and with minimum of attention by staff. In conventional methods and means two types of apparatus are available: the one only provides for auto matic incubation of cultures and determination of the growth at certain intervals; and the other is for automatic injection of sample solution and medium and determination of the growth at a certain time thereafter. The former type suffers from the disadvantages that the sample handling capacity is so limited that it is not possible to simultaneously test a large number of samples.

It is accordingly an object of the present invention to provide a method and an apparatus that overcome the defects inherent in conventional methods and means for shaking culture of microorganisms.

It is another object of the present invention to provide a method and an apparatus that makes a considerable contribution to facilitating and accelerating bacterial growth without loss or leakage of the culture. In addition to increased productivity, the method and apparatus of the invention offer the advantages of easy adaptation to use for different cultures, and rapid adjustability of degrees of shaking. Further, the apparatus of the invention is simple and low-cost.

According to the invention there is provided a frame on which a plurality of holders, wherein test-tubes containing cultures may be held, are rotatably mounted. The frame is mounted on horizontal, parallel bars,and

is freely adjustable forwards or rearwards thereon. The horizontal bars are mounted on vertical support columns, and are freely adjustable upwards or downwards thereon. A horizontal bar is caused to move reciprocally by drive means, and to strike the suspended testtubes at certain points thereon, whereby the test-tubes are caused to shake, and the cultures therein are shaken. The invention offers theadvantages that the effective stroke length of the bar causing test-tubes to shake can be easily and rapidly adjusted, that the shaking frequency of the test-tubes is easily adjusted be adjusting the speed of the bar drive means, and that therefore the exact degree of shaking of the cultures is adjustable and controllable. In addition, the method and apparatus of the invention can provide more vigorous shaking than that achieved in conventional methods and means.

It is a further object of the present invention to provide an automated method and an apparatus for injection of medium, inoculation, incubation with or without shaking, and repeated determination of the growth of bacteria, all in a continuous process, whereby the bacterial growth can be observed in detail and more accurately.

It is a still further object of the present invention to provide a method and an apparatus which makes possible a continuous process wherein sample solutions are diluted, and culture medium and said sample solutions are supplied to test-tubes in an aseptic, constant-temperature chamber, the mixture of sample solution and culture medium is automatically inoculated. said testtubes are transported on a zigzag rail and shaken by a reciprocating bar while being so transported, and the bacterial growth is determined by a turbidimetric method at repeated intervals directly in said test-tubes.

It is a still further object of the invention to provide an apparatus wherein a zig-zag guide rail, the length of which is adjustable, is provided in an aseptic, constanttemperature chamber, test-tubes are freely suspended in holders pivotally mounted on said guide rail, a means for causing said holders to move on said guide rail are provided at certain points adjacentsaid guide rail, a means for injection of sample solutions and culture medium into said freely suspended test-tubes is provided at a station adjacent to said guide rail, said testtubes are shaken by a bar driven by a drive means while being transported on said guide rail, an assessment station, wherein said test-tubes may be halted and the optical density of the cultures therein may be measured in order to determine the bacterial growth, is provided by said guide-rail, whereby test-tubes containing cultures may be transported over a path adjustable length while being shaken and the growth may be determined directly from said test-tubes at variable intervals in a continuous, automatic process.

These and other objectsand features of the present invention will become apparent from the following description taken in conjunction with preferred embodiments thereof with reference to the accompanying drawings in which:

FIG. 1 is a plan view of an apparatus for the shaking of cultures in accordance with one preferred embodiment of the present invention,

FIG. 2 is a side elevational view of the apparatus of FIG. 1,

FIG. 3 is a perspective view of a support frame for a test tube employed in the apparatus of FIG. 1,

FIG. 4 is a perspective view of a holder for a test tube similar to that of FIG. 3, according to another embodiment of the present invention,

FIG. 5 is a front view of a shaking test tube, for purpose of illustrating an experimental method according to the present invention.

FIG. 6 is a graph showing results of an experiment employing the apparatus of FIG. 1 and applying the method of the present invention,

FIG. 7 is a schematic plan view showing a layout of apparatuses for incubation in accordance with another preferred embodiment of the present invention,

FIG. 8 is a cross-sectional view of a drive means 42 employed in the layout of FIG. 7,

FIG. 9 is a plan view of the drive means of FIG. 8,

FIG. 10 is a side-elevational view of a fast advance means 43 employed in the layout of FIG. 7,

FIG. 11 is a plan view of a bar drive means 52 employed in the layout of FIG. 7,

FIG. 12 is a side-elevational view of a means 46 for removing and replacing caps employed in the layout of FIG. 7,

FIG. 13 is a schematic side-elevational view of a supply means employed in the layout of FIG. 7,

FIG. 14 is a plan view of a test-tube holding means 49 employed in the layout of FIG. 7,

FIG. 15 is a cross-sectional view of the holding means of FIG. 14, and

FIG. 16 is a graph showing results of an experiment employing the apparatus of FIG. 7, and applying the method of the present invention.

Before the description proceeds, it is to be noted that like parts are designated by like reference numerals throughout the accompanying drawings for the sake of brevity. It is to be noted that the concept of the present invention as well as its application to equipment for the continuous shaking of a test-tube herein disclosed as a preferred embodiment can be applied in any field of industry other than the medical industry, for example, in connection with liquid, oil or other like fluid if an amount of such fluid is inserted into a bottle or other like receptacles.

Referring first to FIGS. 1 and 2, the apparatus for shaking a culture of micro-organisms to which the present invention is applicable comprises a support base 10, two support columns 11 fixedly implanted in the base 10 near opposite sides thereof, and extending vertically upwards therefrom, and a flat bar 12 is mounted on each support column 11, at right-angles thereto, that is, parallel to the support base 10. The flat bars 12 are also mounted parallel to one another, and extend in the direction of the front of the support base 10. Near one end of each bar 12, there is formed a hole 13, which possesses a diameter slightly larger than that of a support column 11, and permits the bar 12 to be slidably mounted on the corresponding support column 11. In other words, the bars 12 can be moved vertically upwards or downwards to any desired position on the columns 11. Each bar 12 is held in a desired position on the corresponding column 11 by a bolt 14, which screws into a threaded hole 15 formed in the bar end, and contacts the side of the column 11.

A test-tube support frame 16 is mounted on the flat bars 12. The support frame 16 comprises a long main bar 16a, and a plurality of short bars 16b which are formed integrally with the main bar 160, and extend at right-angles thereto. The length of the support frame main bar 16a is approximately equal to the distance between the support columns 11, and each end of the bar 16a has therein a slot 17, into which the flat bar 12 fits slidably, as shown in FIG. 3. In other words, the support frame 16 can be moved horizontally forwards or rearwards along the flat bars 12 to any desired position. The frame 3 is fixed in a desired position by means of bolts 18 each of which fits into a threaded-opening 19 formed in the support frame end, and contacts the side of the corresponding bar 12.

A pin 20 is fixedly attached to and projects from each side of each short bar 1612, near the outer end thereof. that is, the end further removed from the main bar 16a. A hollow, cylindrical holder 21 is provided between each pair of short bars 16!), each holder 21 being mounted on a pair of pins 20 and being free to rotate thereon. A test-tube T provided with a rubber ring 22 is held in each holder 21, the test-tube T being passed through the ring 22 and holder 21, and the test-tube T being supported by the rubber ring 22 in holder 21. The ring 22 being adjustable upwards or downwards, the seating of the test-tube T can be made higher or lower in the holder 21. Also, since each holder 21 is rotatably mounted on pins 20, each test-tube T can be swung forwards or rearwards relative to the main bar 160. Since there is plurality of short bars 16b, and holders 21, a plurality of test-tubes T may be rotatably supported by the support frame 16. Each test-tube T is covered by a cap 23.

A motor 24 is provided on the support base 10. The motor 24 is directly connected to a first wheel 25, which is positioned above the motor 24, and lies parallel to the surface of the base 10. A second wheel 26 level with the first wheel 25 is rotatably mounted on a shaft projecting upwards from the base 10. Power is supplied to the second wheel 26 from the motor 24, for example by a chain sprocket 27, and a chain 28 which transmits drive to another chain sprocket 29 mounted coaxially with the second wheel 26. When, therefore, the motor 24 is actuated, both wheels 25 and 26 are rotated synchronously at the same speed. The description continues below with reference to the first wheel 25and elements associated therewith, it being understood that the description also applies to the second wheel 26. In the upper surface of the wheel 25, there is formed a radial groove 25a, which extends from the center to the periphery of the wheel 25, and accommodates one end of a crankshaft 30. The crankshaft end is slidably mounted in the groove 25a, and is fixed at a desired point therein by a holding pin 31. The stroke of the crankshaft 30 during rotation of the wheel 25 may therefore be varied by adjustment of the position of the crankshaft end in the groove 25a, the stroke being longer the nearer the crankshaft end is to the periphery of the wheel 25. The other end of the crankshaft 30 is rotatably connected to one end of a piston 32 by a connecting pin 117. The piston 32 extends forwards, below the support frame main bar 16a, and is slidably supported by a bearing block 33, which is mounted on the base 10. The piston 32 is caused to slide through the bearing block 33, and is driven in a reciprocating motion together with the crankshaft 30 by rotation of the wheel 25. The other, forward end of the piston has fixedly attached thereto a long bar 34, near one end thereof. The forward end of a second piston 32, which is connected through a second crankshaft 30 and holding pin 31 to the second wheel 26, is fixedly attached to the bar 34, near the other end thereof. The bar 34 is approximately the-same length as the support frame main bar 16a. and contacts the lower portions, that is, below the holders 21, of test-tubes T held in the frame 16. When, therefore, the motor 24 is actuated, the wheels 25 and 26 are rotated, the crankshafts 30 and pistons 32 are driven in a reciprocating motion, the bar 34 is also moved successively forwards and rearwards, and the testtubes T are rocked by the repeated pushes of the bar 34. This rocking of the test-tubes T effectively shakes the contents thereof.

There are no special restrictions concerning the material of the bar 34. The bar 34 is preferably made of a material with a high rigidity, which may be, for example, steel or iron. In consideration of possible damage to test-tubes T, the bar 34 is preferably encased in a tube, of vinyl or similar material. However, such a covering should not be thick, as this lowers the effectiveness of the rocking action by the bar 34.

It is to be noted that, according to the present invention. it is not essential that there be provided a unitary frame for holding test-tubes T, but there may be provided detachable supports 35, as shown in FIG. 4. Each test-tube support 35 comprises a yoke 36, to one side of which a U-or V-shaped forked portion 37 is fixedly attached. The yoke 36 and forked portion 37imay of course be formed integrally. Pins are fixedly attached to the arms of the forked portion 37, near the outer ends thereof. The pins 20 support and permit rotation of a test-tube holder 21. The yoke 36 is hooked onto a rail 39, whereby the holder 21 is mounted in a desired position, and a test-tube T in the holder 21 may be rocked in the same manner as described above. Since each support 35 is a separate unit, yokes 36 may he slid along the rail 39, and test-tubes T may be rocked while being simultaneously transported to some required stage. Also, it is possible to mount a plurality of supports 35 on parallel rails. and test-tubes T supported by the supports 35 on different rails may be simultaneously rocked by connected bars 34.

The means of the invention offers the particular advantage that adjustment of the degree of shaking of the contents of test-tubes T is easily and rapidly effected. The degree of shaking of the contents of test-tubes T depends on the amount of the frequency of rocking of the test-tubes T, and this may be varied by varying the speed of the motor 24, and by adjusting the positions of the crankshaft ends to vary the length of the stroke of the bar 34. Adjustment of the effective stroke length of the bar 34 may also be made by moving the support frame 16 forwards or rearwards on the flat bars 12, or by moving the flat bars 12, and therefore the support frame 16, upwards or downwards on the support columns 11. For example, to obtain the maximum shaking effect, the crankshaft ends are fixed by the holding pins 31 at the outer peripheries of the wheels and 26, the position of the support frame 16 is adjusted so that test-tubes T therein are pushed at the center point of the reciprocal stroke of the bar 34, and the rotational speed of the motor 24 is adjusted, to make the period of the reciprocating motion of the bar 34 double the oscillatory period of the test-tubes T. It is to be noted, however, that, in cases of extreme shaking, if test-tubes T are swung by more than 50, there is a risk of the contents being thrown out therefrom. Therefore, there may be provided a stop 38 fixed on the support base 10 on the opposite side of the test-tubes T from the bar 34, in order to prevent test-tubes T from being swung more than 50. It is, of course, possible to set up a plurality of frames 16 parallel to one another, and to simulta- 6 neously rock test-tubes T in the frames 16 by interconnected bars 34.

Experiments, which are described below, were carried out to compare the effectiveness of the means of the invention with that of conventional shaking culture means.

EXPERIMENT I There were employed uniform test-tubes T having dimensions of 15 X 150 mm and a weight of 15.5 g. In each test-tube T there was placed a culture medium whose composition was comprised of 20 sorbitol, l corn steep liquor and 0.03 calcium carbonate. The medium was inoculated with Acetobacter suboxidans. and the test-tubes were covered by caps having dimensions of 20 X 47 mm and a weight of 6.5 g. The testtubes T were supported at 35 mm from their upper ends by 0 rings attached therearound. and were suspended in holders 21. The test-tubes T were struck cyclically at a point 35 mm from the bottom by an iron bar which was 15 mm in diameter, was covered by a covering of rubber 1 mm thick, and had a stroke length of 40 mm. The impact frequency was varied, that is, the number of cycles per minute of reciprocal motion of the bar was varied, and the rate of sorbose production from sorbitol for 24 hours at 30C, was measured. At the same time the angular displacement 0, as shown in FIG. 5 of the test-tubes T at the different impact frequencies were measured. For the purposes of comparison, cultures comprised of the same constituents were shaken by a conventional, commercially available reciprocal shaker, having a stroke of mm and an im' pact frequency of 149 cpm, for 24 hours at 30C.

The results of the experiment, which are plotted in FIG. 6, showed that sorbose production was accelerated with increased impact frequency up to a peak of about 150 cpm, over which there is a decline. The ratio of sorbose produced to sorbitol added at 150 cpm was 75%, which is an outstandingly marked improvement as compared with a ratio of 40% attainable with the conventional means. It was therefore made clear that far superior results are obtained by the shaking means of the invention.

It was also established that the rate of production of sorbose is in a positive relationship to the angular displacement of the test-tubesT, that is, production increases as the angle of displacement 6 increases. At an impact frequency of 150 cpm, the frequency of reciprocal motion of the bar 34 is exactly twice that of the shaking motion of the test-tubes T, and the bar 34 strikes the test-tubes T at the center of their reciprocal motion (0=0). At this point the momentum of both bar 34 and test-tubes T is maximum. The increased rate of production of sorbose under these conditions may be considered to be due to the fact that the impact imparted to cultures within the test-tubes T is maximum, displacement of test-tubes T is large, and an increased amount of oxygen is transferred into the cultures. The lower rates of sorbose production at impact frequen cies above or below 150 cpm are probably due to the fact that the impact imparted to the test-tubes is less since the bar does not strike the test-tubes T at the center of its reciprocal motion.

EXPERIMENT II The conditions of this experiment were the same as those of Experiment I, except that bars of different materials were used. Results Results No. Bar Material Sorhose Production (7:)

l Bare iron 80 2 Iron covered with rubber I mm thick (Experiment I) 75 3 Iron covered with rubber 3 mm thick 60 As is clear from the above description, the present invention provides a shaking method and means whereby bacterial growth is remarkably increased, without loss or leakage of said cultures.

Referring now to FIGS. 7 to 15, the incubation and determination means according to another embodiment of the invention may be seen to comprise a guide rail 39 housed in a chamber 40, wherein there is maintained as aseptic atmosphere at a constant temperature by a means 41 provided adjacent thereto. The guide rail 39 is a continuous rail having a straight section 39-1 and a series of loops 39a, 39b, 39c, which are generally at right-angles to the straight section 39-1 and each of which comprises two comparatively long, straight, parallel sides.

In this embodiment of the invention, the guide rail 39 has three (first, second and third) loops 39a, 39b, 39c. Needless to say, however, the number of loops may be any number required. At the top end of each loop 39a, 39b and 390, that is, the end thereof further removed from the straight section 39-3, 39-5 39-13, there is provided a drive means 42 on the inside of the curve. The straight section 39-1 joins the bottom end of the outer side of the first loop 39a to the bottom end of the outer side of the third loop 390. A drive means 42 is provided on the inside of the curve where the staight section 39-1 meets the first loop 39a, where the straight section 39-1 meets the third loop 390, and also in approximately the center of the straight section 39-1, that is, generally opposite the bottom end of the second loop 39b. Between the first and second loops 39a and 39b, near the lower ends thereof and on the inside of the curve formed by a guide rail 39-6, which joins the bottom of the inner side of the first loop 39a to the bottom of one side of the second loop 39b, there is provided a fast advance means 43. Another fast advance means 43 is similarly provided between the second and third loops 39 b and 390, on the inside of the curve formed by a guide rail 39-10 joining the bottom of the outer side of the second loop 39b to the bottom of the inner side of the third loop 390. Test-tubes T held' in holders of the same construction as that shown in FIG. 4 of the first embodiment, the yoke 36 of which is slidable on the guide rail 39, are transported, in a manner described below, along the straight section 39-1, up the outer side 39-3 of the first loop 39a, down the inner side 39-5 of the first loop 39a, around the second loop 39b, up the inner side 39-11 of the third loop 390 and down the outer side 39-13 of the third loop 390, and back to the straight section 39-1. In other words the test-tubes T follow a generally zig-zag path. The length of the path over which the test-tubes T are carried may be shortened by providing a guide rail shunt section 44 as shown with a dotted line in FIG. 7 to join the straight section 39-1 directly to the bottom of the second loop 39b, on the side thereof lying nearer the first loop 390. In this case, the test-tubes T are carried half-way along the straight section 39-1 up the shunt section 44,

around the second loop 39!), around the third loop 390, and back to the straight section 39-1. The length of the path over which test-tubes T are transported may be still further shortened by joining the straight section 39-1 directly to the bottom of the inner side of the third loop 39c by a shunt section 45 as shown with a dotted line in FIG. 7. In this case, test-tubes T simply travel around the third loop 39:. Whatever the length of the path over which test-tubes T are transported, the testtubes T are initially brought to a supply station I, and finally brought to an assessment station II. The supply station I is situated generally opposite the bottom end of the third loop 39c, and comprises a means 46 for removing and replacing caps, a means 47 and 48 for supplying culture medium, sample solutions, inoculum and any other required substances, into test-tubes T. The assessment station II is situated opposite the bottom of the outer side of the third loop 396, and comprises a test-tube holding means 49, an electrophotometer 50, and a recording unit 51. Parallel to the straight side portions of each loop 39a, 39b and 39c there are provided reciprocating long bars 34a. That is, there is one pair of bars 34a in association with each loop 39a, 39b and 390, the bars 34a in each pair being on opposite sides of the corresponding loop 39a, 39b and 39 c. The bar 34a facing the outer side of the third loop 390 is divided into two independent sections 34b and 340, the bar section 34b being longer and facing the upper portion of the outer side of the third loop 39c, and the bar section 340 being shorter and facing a lower portion thereof. A single reciprocating long bar 34d is provided parallel to the guide rail straight section 39-1. Each pair of bars 34a and 34b and 34c, is indirectly supported by, and driven by a drive means 52, which is located inside the corresponding loop 39a and 39b and the single bar 34d is driven through a linkage 53 thereto. The short bar 340 is driven by an independent drive means 74.

The above-mentioned holders 35, drive assemblies 52, bars 34, drive means 42, and fast advance means 43 will be described in further detail hereinbelow. Unless otherwise specified, the description will be in reference to single means 35, 42, 43, or set of means 52, 34, it being understood that there is a plurality of indentical means 35, 42, 43, or set of means 52, 34.

Referring now to FIGS. 4 and 8, the holder 35 may be seen to comprise a yoke 36, and a forked section 37, which is integral with and generally at right-angles to one side of the yoke 36. On the other side of the yoke 36 there is fixedly attached a projecting stud 54, which is engaged by drive means 42, or fast advance means 43, as described below. The yoke 36 is hooked onto the guide rail 39, whereby the holder 35 is slidably supported thereby. In this configuration, the forked section 37 is generally horizontal. A pin 20 is provided at the end of each arm of the forked section 37. A hollow cylindrical support 21 is rotatably mounted on the pins 20, and between the arms of the forked section 37. On the upper portion of the test tube T there is provided a rubber ring 22, the setting of which relative to the support 21 is adjustable upwards or downwards, and which acts as a support pad for the upper portion of the cylindrical support 20. A test-tube T to be transported around the guide rail 39 is slid downwards through the ring 22 and-hollow cylindrical support 21, and is held suspended thereby. The amount by which the rubber ring 22 stands clear of the cylindrical support 21 being adjustable, the setting of the test-tube T may be adjusted upwards or downwards relative to the holder 35, and hence relative to the guide rail 39. When the holder 35 is in repose on the guide rail 39, the testtube T is held suspended in a vertical position. However, since the cylindrical support 21 may pivot on the pins 20, and swing freely in the arms of the forked section 37, the test'tube T also may be swung freely, towards or away from the guide rail 39.

In FIG. 7 there are shown only a few test-tubes T suspended in holders 35 hooked on the guide rail 39. In actual use of the automated culture means of the invention there are test-tubes T in holders 35 over the whole length of the guide rail 39, except for the portions 39-6 and 39-10 thereof facing the fast advance means 43. When being transported around the guide rail 39, testtubes T contain bacterial cultures supplied thereinto at the supply station I. Since it may not be possible to maintain a completely aseptic atmosphere in the chamber 40, and there may be remnant bacteria, even in asceptic air supplied by the means 41, and since such remnant bacteria could contaminate cultures contained in the test-tubes T, especially if the cultures are of low activity, each test-tube T is covered by a cap 23, to further protect the contents thereof.

Referring to FIG. 8, the drive means 42 comprises a horizontal wheel 55, which rotates about a vertical axis 56, and which is driven by a rotary solenoid 57. Indentations 55a are formed at even intervals in the outer periphery of the wheel 55a. The indentations 55 engage studs 54 projecting from the sides of yokes 36 of the holders 35, whereby rotation of the wheel 55 may cause holders 35 to be driven around curved portions of the guide rail 39. For example, referring more particularly to FIG. 7, the wheel 55 at the top of the first loop 39a follows the curve of the guide rail 39-4 over a semicircular arc. When, therefore, a holder 35 carrying a test-tube T comes to the top of the outer side of the first loop 39a, the stud 54 on the yoke 36 thereof is engaged by an indentation 55a in the wheel 55 at the top of the first loop 39a, and the rotary solenoid 57 being actuated and the wheel 55 rotated, the holder 35 is carried 180 around the top end of the first loop 39a, and to the top of the inner side thereof. When the holder 35 is thus carried around the top of the first loop 39a it pushes the preceding holder 35 forwards, and is itself subsequently pushed forward by the succeeding holder 35 carried around the top of the first loop 39a by engagement of the stud 54 thereof by the succeeding indentation 550 of the wheel 55. Holders 35 are similarly carried through 180 around the tops of the secnd and third loops 39b, 39c successive holders 35 pushing preceding holders 35 forwards. The wheels 55 of the drive means 42 provided at the curved sections where the guide rail straight section 39-1 meets the first and third loops 39a and 390 follow the curve of the guide rail 39-2 and 39-14 through 90 only. At these points thereof, holders 35 are moved forwards in the same manner as described above. If, however, a shunt section 45 is installed between the straight section 39-1 and the third loop 39c, the wheel 55 at that location carries holders 35 through 180, from the bottom of the outer side of the third loop 390 to the bottom of the inner side thereof. The wheel 55 of the drive means 42 provided by the guide rail straight section 39-1, at the bottom of the second loop 39b, normally advances holders 35 in a'straight line only, along the straight section 139-]. If. however. a shunt section 44 is installed between the straight section 39-1 and the second loop 10 3%, the wheel 55 at that location carries holders 35 though from the straight section 39-1, onto the shunt section 44, successive holders 35 pushing preceding holders 35 upwards on the second loop 3%. All the drive means 42 are actuated synchronously, whereby test-tubes T are transferred along the guide rail 39 at the same speed at each location of a drive means 42.

As shown in FIGS. 9 and 10 the fast advance means 43 comprises a cylinder 58 and plunger 59 assembly, and a rotary solenoid 60. The cylinder 58 and plunger 59 assembly is positioned vertically, and is connected to, and rotated in a horizontal plane by the rotary solenoid 60, which is provided therebelow. The outer end of the plunger 59 is fixedly connected to the end of one section of a bracket-like arm 61. The arm 61 comprises a vertical section 61a, connected to the plunger 59, a horizontal section 61b, and a vertical section 610. The section 61c extends vertically downwards, and on the inside of the bottom end thereof there is formed a groove 62. The groove 62 is for engagement of studs 54 on holders 35. The arm 61 being connected to the plunger 59, when the cylinder 58 and plunger 59 assembly is actuated to raise or lower the plunger 59, the arm 61 is likewise raised or lowered. Also, when the rotary solenoid 60 rotates the cylinder 58 and plunger 59 assembly, the arm 61 is likewise rotated in a horizontal plane.

In the starting position of, for example the, fast advance means 43 between the first and second guide rail loops 39a and 391), the plunger 59 and arm 61 are raised, and the arm section 61c lies over point A, that is, the start of the curved section 39-6 between the first and second loops 39a and 39b. When a holder 35 pushed by other holders 35 comes to point A. the cylinder 58 and plunger 59 assembly is actuated to retract the plunger 59 and lower the arm 61, and the groove 62 of the section 61c slides onto, and engages the stud 54 of the holder 35. Next, the rotary solenoid 60 is actuated to swing the cylinder 58 and plunger 59, and arm 61 through so that the arm end section 610 goes from point A to point B. Engagement of the groove 62 with the holder stud 54 causes the holder 35, and testtube T held thereby, to be similarly swung along the curve A-B. When the holder 35 is brought to point B it pushes the preceding holder 35 forward. The cylinder 58 and plunger 59 assembly is now actuated to push the plunger 59 upwards, whereby the arm 61 is raised, and the groove 62 disengages from the stud 54. Next, the solenoid 60 swings the arm 61 back through 180, to bring the section 610 back to point A, ready to engage the next holder 35, which has been pushed forward to point A. The above-described operation is then repeated, and this next holder 35 is transferred rapidly to point B. Holders 35 are transferred in the same manner and at the same speed over the curved section A-B of the curved section 39-10 between the second and third loops 39b, 390.

The operation of the drive means 42 and fast advance means 43 are adjusted and synchronized so that the time required for transfer of holders 35 over all curved sections 39-2, 39-4, 39- 14, of the guide rail 39 is the same, whereby a smooth flow is maintained. The rate of advance of test-tubes T is also such that test-tubes T may be halted for 30 second intervals, to permit injection of sample solution and inoculation thereinto at the supply station I, and determination of the growth of bacteria atthe assessment station [1.

Referring to FIGS. 8 and 11. the drive means 52 for actuating long bars 34 may be seen to comprise connecting rods 63, crank-shafts 64, and horizontal wheels 65. The connecting rods 63 are horizontally and slid ably supported in slide bearing blocks 66. which are fixedly mounted on the base of the chamber 40. Opposite ends of the rods 63 are fixedly attached to bars 34a on opposite sides of a guide rail loop 39, the connecting rods 63 being generally at right-angles to the bars 340. The connecting rods 63 slide in the bearing blocks 66 and permit the bars 34a to be moved towards or away from the guide rail 39. Also, because of the connecting rods 63, movement of one bar 340 causes the other bar 34a to move in the same manner. The ends 64a of two crank-shafts 64 are rotatably attached to one bar 34a. The other ends 64b of the crankshafts 64 are rotatably attached to wheels 65 by holding pins 67. Rotation of the wheels 65 therefore causes the crank-shafts 64, and bar 34a attached thereto, to move in a reciprocating motion. Each pin 67 is adjustably fixable in a radial groove 68 formed in the upper surface of a wheel 65. In other words, the end 64a of each crankshaft 64 may be moved to, and fixed at different locations between the center and the periphery of the corresponding wheel 65, whereby the length of the stroke of the crankshaft 64, and hence bar 34a, may be varied. One wheel 65 is fixedly mounted on and driven by the drive shaft 69 of a motor 70. The other wheel 65 is fixedly mounted on a rotatable shaft 71, which is mounted vertically on the base of the chamber 40, and on which a chain sprocket 72 is mounted. A chain sprocket 72 is also mounted on the motor drive shaft 69. The chain sprockets 72 engage a chain 73 connecting the drive shaft 69 and shaft 71. Therefore, when the drive shaft 69 is rotated, the crank-shafts 64, and the bar 34a attached thereto are driven reciprocally, the bar 34a being moved towards or away from the guide rail 39. When the bar 34a is thus driven reciprocally, it cyclically strikes the lower portions of test-tubes T suspended in holders 35 on one side of the loop 39, whereby the test-tubes T are caused to shake, and the cultures therein are shaken, and the supply of oxygen thereto is increased. The degree of shaking of cultures in test-tubes T may be varied by adjustment of the stroke length of the bar 34a, that is, by fixing crank-shaft ends 64b nearer or further away from the peripheries of wheels 65, to lengthen or shorten the stroke of the crank-shafts 64. The bar 34a attached to the crankshaft ends 64a being driven reciprocally, the bar 34a on the other side of the loop 39 is similarly driven, because of the connection of the rods 63 sliding in the bearings 66, and this bar 34a also strikes test-tubes T in the same manner, and producing the same results.

Other bars 34 associated with other guide rail loops 39, including the bar sections 34b of the third loop 39c, or the bar 34d opposite the guide rail straight section 39-1, are similarly supported, connected and driven by connecting rods 63 sliding in bearing blocks 66 and crank-shafts 64 driven reciprocally by wheels 65. Chain sprockets 72 are mounted on the rotary shafts 71 of all wheels 65, and are engaged, and connected by chains 73. All wheels 65 are therefore driven synchronously by the motor 70, whereby all bars 34 are driven in synchronous reciprocal motion so that all test-tubes T on the guide rail 39 are caused to shake with the same frequency. The short bar section 340 on the path of test-tubes T immediately before the assessment station II is further provided with an independent drive means 12 74, whereby the bar section 340 may be driven in reciprocal motion even when the bar section 34b and other bars 34a are undriven and remain stationary.

It is to be noted that various modes of operation are possible with the apparatus of the invention. Test-tubes T containing cultures may be transported around the guide rail 39 by the action of the drive means 42 and fast advance means 43, and simultaneously caused to shake by the bars 34. Alternatively, the action of the drive means 42 and fast advance means 43 may be stopped and only the bars 34 be actuated, whereby test tubes T are shaken, while test-tubes T remain in the same locations. Also if the short bar section 340 only is actuated, test-tubes T may be transported around most of the guide rail 39 without being shaken, and then be agitated for a short time only immediately prior to coming to the assessment station II, whereby bacterial cells in the test-tubes T are evenly mixed.

Referring to FIG. 12, the means 46 for removing and replacing caps 23 comprises an electrically-driven cylinder 75 and plunger 76 assembly, and a rotary solenoid 77. The cylinder 75 and plunger 76 assembly is positioned vertically, and is connected to, and rotated in a horizontal plane by the rotary solenoid 77, which is provided therebelow. The outer end of the plunger 76 is fixedly connected to the end of one section of a bracket-like arm 78. The arm 78 comprises a vertical section 78a, connected to the plunger 76, a horizontal section 7812, and vertical section 780. The vertical section 780 extends vertically downwards, and at the outer end thereof thereof there is provided an electromagnet 79 and a grab 80, which is controlled by the electromagnet 79. When a test-tube T in a holder 35 is brought to the position of the cap 23 removal and replacement means 46, it comes under the grab 80. The cylinder 75 and plunger 76 assembly is then actuated to draw the plunger 76 downwardsand into the cylinder 75, thus lowering the arm 78 and grab 80. When the grab has been lowered to a position around the cap 23 on the test-tube T, the electromagnet 79 is actuated to close the grab 80, which therefore grips the cap 23. While the grab 80 continues to grip the cap 23, the cylinder 75 and plunger 76 assembly is actuated to raise the plunger 76 and arm 78, whereupon the cap 23 is removed from the test-tube T by the grab 80. The rotary solenoid 77 is now actuated to rotate the cylinder 75 and plunger 76 assembly and arm 78, and swing the cap 23 out of alignment with the test-tube T. Culture medium. sample solution and inoculum are now supplied into the test-tube T by the supply means 47 and 48, as described below. When this supplying process is completed, the rotary solenoid 77 is again actuated to rotate the cylinder 75 and plunger 76 assembly and arm 78, and swing the cap 23 back into alignment with the test-tube T. Next the cylinder 75 assembly is actuated to move the plunger 76 and arm 78 downwards. and bring the cap 23 back onto the test-tube T. After this, the electromagnet 79 opens the grab 80, which therefore releases the cap 23, and then, the plunger 76, arm 78, and grab 80 are raised vertically, in readiness for the next test-tube T, and the cap 23 is left on the first test-tube T, to protect the culture therein.

When a cap 23 is removed from a test-tube T, as described above, requisite materials are supplied into the test-tube T by the above-mentioned supply means 47 and 48, which are shown in FIG. 13. The supply means 47 and 48 comprise an electrically driven cylinder 81 and plunger 82 assembly, and a rotary solenoid 13 83. There are also separately prepared supplies of culture medium 84, inoculum 85, and water 86. The cylinder 81 and plunger 82 assembly is positioned vertically, and is connected to, and rotated in a horizontal plane by the rotary solenoid 83, which is provided therebelow. One end 87a of an arm 87 is fixedly attached to the outer end of the plunger 82. The arm 87 is aligned horizontally, and the other end 87b thereof supports nozzles 88 and 89, which are directed vertically downwards, the nozzle 88 being shorter than the nozzle 89. When the rotary solenoid 83 rotates the cylinder 81 and plunger 82 assembly, the arm 87 is likewise swung in a horizontal plane. The arm 87 is swung so that the outer end 8719 thereof, and nozzles 88 and 89, supported thereby. come to one of two positions C and D. In position C, as shown with solid lines in FIG. 13, the arm end 87b and nozzles 88 and 89 are vertically above a sample solution container 90 in a sampling unit 91. The sampling unit 91 contains a plurality of containers 90, which are moved successively into alignment with nozzles 88 and 89 in position C. Each container 90 holds sample solution 92, and is covered with a sheet of aluminum foil 93. In position D, as shown with dotted lines in FIG. 13, the arm end 87b and nozzles 88 and 89 are vertically above a test-tube T, from which the cap 23 has been removed.

The nozzles 88 and 89 are connected by flexible supply lines 94 and 95 to the supply means 47 and 48, respectively. The supply means 47 includes an injector 96 comprising two microcylinders 97 and 98, which are connected to branch lines of the supply line 94. The microcylinder 97 is also connected to the culture me dium 84, and the microcylinder 98 is connected to the inoculum 85. The supply means 48 includes an injector 99 comprising two microcylinders 100 and 101, which connect to the supply line 95. The microcylinder 101 also connects to the supply of water 86.

When a test-tube T is at supply station I, and while the cap 23 is being removed therefrom, the arm 87 is raised and is at position C, above a container 90, which holds sample solution 92 and is covered by a sheet of aluminium foil 93. The cylinder 81 and plunger 82 assembly is actuated to retract the plunger 82, and lower the arm 87. Hereupon, the longer nozzle 89 pierces the aluminium foil 93 and the lower end thereof enters the sample solution 92. The microcylinders 100 and 101 are now actuated independently, the microcylinder 100 drawing in a required amount of sample solution 92, and the microcylinder 101 similarly drawing in water 86. At the same time the cylinder 97 and microcylinder 98 also are actuated independently, drawing in required amounts of culture medium 84 and inoculum 85, respectively. Next, the cylinder 81 and plunger 82 assembly is actuated to raise the plunger 82 and arm 87, thus withdrawing the nozzle 89 from the sample solution container 90, whereupon the sampling unit 91 moves the next container 90 into line. While the arm 87 is still raised, the rotary solenoid 83 is actuated, and the arm 87 is swung to position D. The plunger 82 is then retracted, to lower the arm 87, and bring the nozzles 88 and 89 into the mouth of the test-tube T. The contents of the microcylinder 100, and cylinder 101 are now injected together along the supply line 95, and the contents of the cylinder 97 and microcylinder 98 are injected together along the supply line 94, whereby the required amounts of sample solution 92, water 86, culture medium 84, and inoculum 85 are injected into the test-tube T. The arm 87 is then raised 14 and swung back to position C, the cap 23 is replaced on the test-tube T, the test-tube T is moved forwards, and the next testtube T is brought into line, and the abovedescribed process is repeated.

To prevent contamination by external air during this injection process, the section containing sample solutions is further protected by a plastic cover. and also aseptic air supplied to the chamber 40 by the aseptic constant supply means 41 keeps the pressure inside the chamber 40 positive with respect to outside pressure. An aseptic atmosphere may be further ensured by the provision of glow-type germicide lamps, or gas means employing ethylene oxide for eliminating bacteria, or similar means.

As shown in FIGS. 14 and 15 the above-mentioned test-tube holding means 49 in the assessment station 11 comprises an electrically driven cylinder 102 and plunger 103 assembly, a clamp shift element 104, clamps 105, and a fixed stand 106.

The fixed stand 106 comprises a top plate 106:: and a lower plate 1061), both of which are horizontal and supported by vertical supports 1060. Two elliptical openings 107 are formed in the top plate 106a. The major axes of the openings 107 lie on the same line. Two corresponding elliptical openings 107 are formed in the lower plate 106b, in line with the openings 107 in the top plate 106a. Each clamp 105 comprises an upper, semicircular portion 105a and a lower, rod portion 105b. Each clamp rod portion 105]) passes through the fixed stand top and lower plates 106a and 10612, and fits slidably in a pair of openings 107 formed therein. The lower end of each portion 105]? extends to below the lower plate 106b, and a retainer ring 108 is attached thereto, to prevent the clamp 105 being slid upwards out of the fixed stand 106. When the clamps 105 are thus slidably mounted in the stand 106, the lower ends of the clamp semicircular portions 105a rise slidably on the upper surface of the fixed stand top plate 106a, and the semicircular portions 105a lie opposite, and are curved towards one another. In each clamp semicircular portion 1050 there is formed an opening 109, which permits passage of light for turbidimetric determination of bacterial growth in test-tube T, as described below. The clamps 105 may be moved towards or away from one another by the sliding motion of the rod portions 105]) in the openings 107.

The clamp shift element 104 has a substantially trapezoidal shape, and has two longitudinal guide slots 110 which are inclined at an angle to one another, whereby the guide slots 110 form a general V shape. The clamp shift element 104 slides freely between the top and lower plates 106a and 106b of the fixed stand 106, and in this configuration the clamp rod portions 105b lie within the guide slots 110. When therefore the clamp shift element 104 is pushed towards the fixed stand 106, and the narrow end of the V formed by the guide slots 110 approaches the stand 106, the clamp rod portions 105!) are compelled to move along the guide slots 110, and also along the openings 107, towards one another, that is, the clamps 105 are brought together. Contrariwise, when the clamp shift element 110 is pulled away from the stand 106, the clamps 105 are moved apart.

The clamp shift element 104 is caused to move towards or away from the stand 106 by actuation of the electrically driven cylinder 102 and plunger 103 assembly, the line of action of which is generally parallel to the guide rail 39. Opposite ends of a connecting shaft 111 are fixedly attached to the plunger 103 and to the narrow end of the shift element 104, respectively. Outward or inward movement of the plunger 103 with respect to the cylinder 102 therefore causes the shift element 104 to move towards or away from the stand 106, and the clamps 105 to move together or apart. As described earlier, the assessment station II is located near the lower end of the outer side of the third guide rail loop 390, and below the reciprocating bar short section 34c. When a test-tube T transported on the guide rail 39 travels down the outer side of the third loop 340, and past the short bar section 340, it comes to a position above the fixed stand 106 and between the clamps 105. The cylinder 102 and plunger 103 assembly is thereupon actuated to cause the plunger 103 to move outwards, the clamps 105 are moved together, and the clamp semicircular portions 105a fit around, and secure the test-tube T in a fixed position. When the test-tube T is thus secured it still remains in its holder 35.

The above-mentioned electrophotometer 50 comprises a light source 112, lens assembly 113, and slit board 114, which are located on one side of the fixed stand 106, and a photoelectric cell 116, which is located on the opposite side of the fixed stand 106. The lens assembly 113 is situated between the light source 112 and the stand 106, and the principal focal plane thereof lies generally in line with the center of the stand 106, that is, where a test-tube T is held. The board 114 has a slit 115 formedtherein, to permit passage of light from the light source 112, and is provided within the lens assembly 113. When a test-tube T is held in the clamps 105, as described above, light from the light source 112 is directed by the lens assembly 113 and slit 115 through the opening 109 in one clamp 105a, through the culture contained in the test-tube T, through the opening 109 in the other clamp 105a, and onto the photoelectric cell 1 16. The optical density of a culture is proportional to the growth of bacteria therein, and the value obtained is recorded immediately in the recording unit 51. There is, of course, no need for intervention of staff to determine and record this value, but the whole process is effected automatically by suitable electrical connections between the photoelectric cell 116 and recording unit 51.

When this process is completed, the plunger 103 is drawn in, the clamp shift element 104 is drawn away from the stand 106, and the clamps 105 release the test-tube T. The test-tube T is now pushed forward by the next test-tube T moving down the outer side of the 3rd loop 39c, after which this next test-tube T is held in the clamps 105 and the optical density of the light by the culture therein determined and recorded, as described above. Alternatively, the first test-tube T may be removed. In either case, succeeding test-tubes T are brought to the assessment station II and the growth of the cultures therein determined and recorded.

Thus, according to the present invention, testtubes T containing bacterial cultures are held in holders and advanced one step at a time by drive means 42 and fast advance means 43 along a guide-rail 39 in a chamber wherein an aseptic atmosphere at constant temperature is maintained by the means 41. The length of the guide rail 39 is adjustable, and while test-tubes T are' transported thereon they may be shaken by reciprocating long bar 34. Successive test-tubes T are brought to a supply station I, where culture medium sample solution and inoculum are automatically supplied thereto, and are subsequently brought to an assessment station 16 II, where the bacterial growth in culture therein is automatically determined and recorded.

Experiments were carried out to assess the operational efficiency of the method and means of the invention.

EXPERIMENT III 200 test-tubes, having dimensions of 15 X 150 mm, and a weight of 15.5 i 0.2 g were employed. An O-ring was provided around each test tube at the site of l 15 mm for the bottom thereof 3 ml of a culture medium. having a composition comprising 20% sorbitol, 1% corn steep liquor and 0.03% calcium carbonate, were inserted in each test-tube, and protected by a rubber cap, having dimensions of 20 X 47 mm, and a weight of 6.5 g, covering the test-tube. The culture medium in each test-tube was innoculated with Acetobactor suboxidans, and the experiment proceeded with the production of sorbose, at 30C, for 24 hours, during which time the test-tubes were shaken by a reciprocating long bar striking the bottoms thereof, the impact frequency being 150 cycles per minute (cpm), and the bar having a stroke length of 40 mm. For the purposes of comparison, conventional shaking equipment, having an impact frequency of cpm, and a stroke length of 35 mm, was employed for the production of sorbose under the same conditions, that is, at 30C for 24 hours. The results of the experiment show that whereas the progression rate of sorbose production from sorbitol using conventional equipment was only 40%, the average production rate attained with the means of the invention was 80%, thus demonstrating the marked superiority of the means of the invention. It is also to be noted that the coefficient of variation from this average value for 200 samples was only i 3%, in other words the rate of sorbose production is extremely even and reliable.

EXPERIMENT IV There were employed test-tubes T having dimensions of 15 X mm, and being of equal thickness. The empty test-tubes were covered with rubber caps, and after sterilization were suspended in holders in an aseptic, constant-temperature chamber, which was set to 37C. L-isoleucine solutions varying from 0 to 150 ,ug/ml in concentration were pre-loaded into sample solution containers, the samples being divided into 8 levels of 10 samples each. The containers were covered with aluminum foil, and, after sterilization, were mounted in a sampling unit. There was also prepared water for dilution of samples and a suspension of Leuconostoc mesenteroides in the medium for isoleucine assay. Operation then commenced, 0.1 ml of the sample solution being drawn into one microcylinder of one injector and 1.4 ml of water being drawn into the other cylinder thereof, while at the same time 1.5 ml of the culture medium containing the inoculum suspension was drawn into the cylinder of another injector. Suc cessive test-tubes were brought into line with the injector nozzles, the caps removed therefrom, the abovedescribed quantities of sample solution, culture medium and inoculum injected there into, and then the caps were replaced thereon. The test-tubes were slid onto the guide rail at 30 second intervals, and then left stationary, the cultures therein being allowed to incubate for approximately 24 hours. After this period, the test-tubes were shaken by the shaking having the short bar means to produce a uniform suspension of bacteria,

17 and then the test-tubes were brought one at a time to the assessment station, where they were held stationary and the optical density at 660 mp. was measured. The results obtained are shown in the graph of FIG. 16, in which, for comparison, are also plotted the results obtained by using a conventional manual method.

The results show that a concentrations of and 20 1.1. g/ml, the coefficient of variations with the conventional method were 1*: 1.3 and i 0.8, respectively, whereas the variations using the means of the invention were only i 0.6 and i 0.4.

As is clear from the above description, the present invention provides a method and means wherein the whole process, from injection of sample solution, culture medium, and inoculum into test tubes to the repeated measurement of bacteria growth in the cultures, can be carried out automatically with improved accuracy. The invention also offers the advantages of improved efficiency and economy of space by carrying test-tubes containing cultures over a zig-zag path. The construction of the zig-zag guide rail supporting the test-tubes makes it possible to adjust the length of the path over which the test-tubes are carried, and so to adjust the number of test-tubes dealt with, and vary to the duration of the process. The degree of shaking of test tubes is easily adjustable by varying the impact frequency or the stroke length of the reciprocating bar which strikes the test-tubes containing the cultures, or by adjusting the setting of the test-tubes in the holders upwards or downwards. The method and means of the invention also offer great advantages over conventional methods and means with regards to the measurement of bacterial growth in the cultures. With conventional methods and means, it is necessary to transfer the culture of each test tube into the cell in an electrophotometer. There is a possibility of contamination with other cultures during this transfer process. Conventional methods and means have the further disadvantages that determination of bacterial growth by the turbidimetric method can be effected only once for each culture. In contrast to this, with the method and means of the invention, the optional density of cultures may be determined without transfer of the cultures into the cells, and the determination is therefore simple and may be carried out repeatedly if so required. The invention also offers the advantage that portions of the cultures may be withdrawn at any stage in the incubation thereof, reversing the action of the injection nozzles at the supply station.

What is claimed is:

l. A method for bacterial culture wherein sample solution, culture medium and inoculum are placed in a plurality of test tubes, said test tubes are suspended from a horizontal axis above the center of gravity thereof, and said test tubes are cyclically struck by a reciprocating bar acting on a certain level, whereby an oscillatory motion is imparted to said test tubes, and said test tubes and the contents thereof are shaken.

2. A method as claimed in claim 1 wherein the point at which the test tubes are struck and the frequency of impact is such that the period of the oscillatory motion of the test tubes is half the frequency of impact and the amount of oscillatory motion is no greater than 50 from the vertical.

3. A method for continuous bacterial culture and the determination of the growth thereof. wherein sample solution, culture medium, and inoculum are automatically supplied to test tubes freely suspended inholders which are rotatable around a horizontal axis, the holders being supported by and slidable on a guide rail within an aseptic, constant temperature chamber, sliding said holders holding said test tubes around said guide rail by suitable drive means, cyclically striking said test tubes by striking there against at least one bar by moving the bar reciprocally on a certain level for imparting an oscillatory motion to said test tubes and shaking the contents thereof, and repeatedly determining the bacterial growth in said test tubes at a certain location along said guide rail without removal of said holders from said guide rail or removal of said contents from said test tubes.

4. An apparatus for bacterial cultures by shaking comprising a support means, a frame mounted on said support means for free adjustment upwards, downwards, fowards and rearwards relative to the support means, test tube holders mounted on said frame for oscillating movement around a horizontal axis in said frame, for holding test tubes suspended therefrom which test tubes contain sample solution, culture medium and inoculum to be incubated, whereby said test tubes may oscillate freely about the points of support of said holders, a drive means and a bar connected to said drive means and driven in a continuous reciprocating motion towards and away from the positions of the test tubes suspended in said holders for cyclically striking said test tubes, whereby the test tubes are caused to oscillate and contents thereof are shaken.

5. An apparatus for continuous bacterial culture comprising a zig-zag guide rail, an aseptic constant temperature chamber within which said guide rail is mounted, test tube holders slidably positioned on said guide rail, said holders being oscillatable around a horizontal axis for permitting test tubes suspended therefrom to oscillate freely, advance means provided at suitable locations adjacent to said guide rail for advancing said holders along said guide rail one at a time and at regular intervals, means adjacent one point along said guide rail for automatic supply of sample solution and culture medium to said test tubes and for injection of said sample solution and culture medium with inoculum, a holding station at a further point along said guide rail having means for holding test tubes stationary and determining the bacterial growth in the test tubes without removal of said contents from said test tubes, bars extending parallel to certain portions of said guide rail, and drive means connected to said bars for driving said bars in a reciprocating motion along a certain level for cyclically striking test tubes freely suspended in said holders along said guide rail, whereby contents of said test tubes are shaken and bacterial growth therein is accelerated. 

1. A method for bacterial culture wherein sample solution, culture medium and inoculum are placed in a plurality of test tubes, said test tubes are suspended from a horizontal axis above the center of gravity thereof, and said test tubes are cyclically struck by a reciprocating bar acting on a certain level, whereby an oscillatory motion is imparted to said test tubes, and said test tubes and the contents thereof are shaken.
 2. A method as claimed in claim 1 wherein the point at which the test tubes are struck and the frequency of impact is such that the period of the oscillatory motion of the test tubes is half the frequency of impact and the amount of oscillatory motion is no greater than 50* from the vertical.
 3. A METHOD FOR CONTINUOUS BACTERIAL CULTURE AND THE DETERMINATION OF THE GROWTH THEREOF, WHEREIN SAMPLE SOLUTION, CULTURE MEDIUM, AND INOCULUM ARE AUTOMATICALLY SUPPLIED TO TEST TUBES FREELY SUSPENDED INHOLDERS WHICH ARE ROTATABLE AROUND A HORIZONTAL AXIS, THE HOLDERS BEING SUPPORTED BY AND SLIDABLE ON A GUIDE RAIL WITHIN AN ASPETIC, CONSTANT TEMPERATURE CHAMBER, SLIDING SAID HOLDERS HOLDING SAID TEST TUBES AROUND SAID GUIDE RAIL BY SUITABLE DRIVE MEANS, CYCLICALLY STRIKING SAID TEST TUBES BY STRIKING THERE AGAINST AT LEAST ONE BAR BY MOVING THE BAR RECIPROCALLY ON A CERTAIN LEVEL FOR IMPARTING AN OSCILLATORY MOTION TO SAID TEST TUBES AND SHAKING THE CONTENTS THEREOF, AND REPEATEDLY DETERMINING THE BACTERIAL GROWTH IN SAID TEST TUBES AT A CERTAIN LOCATION ALONG SAID GUIDE RAIL WITHOUT REMOVAL OF SAID HOLDERS FROM SAID GUIDE RAIL OR REMOVAL OF SAID CONTENT FROM SAID TEST TUBES.
 4. An apparatus for bacterial cultures by shaking comprising a support means, a frame mounted on said support means for free adjustment upwards, downwards, fowards and rearwards relative to the support means, test tube holders mounted on said frame for oscillating movement around a horizontal axis in said frame, for holding test tubes suspended therefrom which test tubes contain sample solution, culture medium and inoculum to be incubated, whereby said test tubes may oscillate freely about the points of support of said holders, a drive means and a bar connected to said drive means and driven in a continuous reciprocating motion towards and aWay from the positions of the test tubes suspended in said holders for cyclically striking said test tubes, whereby the test tubes are caused to oscillate and contents thereof are shaken.
 5. AN APPARATUS FOR CONTINUOUS BACTERIAL CULTURE COMPRISING A ZIG-ZAG GUIDE RAIL, AN ASEPTIC CONSTANT TEMPERATURE CHAMBER WITHIN WHICH SAID GUIDE RAIL IS MOUNTED, TEST TUBE HOLDERS SLIDABLY POSITIONED ON SAID GUIDEE RAIL, SAID HOLDERS BEING OSCIL LATABLE AROUND A HORIZONTAL AXIS FOR PERMITTING TEST TUBES SUSPENDED THEREFROM TO OSCILLATE FREELY, ADVANCE MEANS PROVIDED AT SUITABLE LOCATIONS ADJACENT TO SAID GUIDE RAIL FOR ADVANCING SAID HOLDERS ALONG SAID GUIDE RAIL ONE AT A TIME AND AT REGULAR INTERVALS, MEANS ADJACENT ONE POINT ALONG SAID GUIDE RAIL FOR AUTOMATIC SUPPLY OF SAMPLE SOLUTION AND CULTURE MEDIUM TO SAID TEST TUBES AND FOR INJECTION OF SAID SAMPLE SOLUTION AND CULTURE MEDIUM WITH INOCULUM, A HOLDING STATION AT A FURTHER POINT ALONG SAID GUIDE RAIL HAVING MEANS FOR HOLDING TEST TUBES STATIONARY AND DETERMINING THE BACTERIAL GROWTH IN THE TEST TUBES WITHOUT REMOVAL OF SAID CONTENTS FROM SAID TEST TUBES, BARS EXTENDING PARALLEL TO CERTAIN PORTIONS OF SAID GUIDE RAIL, AND DRIVE MEANS CONNECTED TO SAID BARS FOR DRIVING SAID BARS IN A RECIPROCATING MOTION ALONG A CERTAIN LEVEL FOR CYCLICALLY STRIKING TEST TUBES FREELY SUSPENDED IN SAID HOLDERS ALONG SAID GUIDE RAIL, WHEREBY CONTENTS OF SAID TEST TUBES ARE SHAKEN AND BACTERIAL GROWTH THEREIN IN ACCELERATED. 